This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-GM-101: Structural analysis of macromolecular complexes. The focus of this proposal is the development and optimization of a novel approach for high-resolution direct imaging of biological processes as they occur in real-time. The "live" movies will be acquired using a Dynamic Transmission Electron Microscope that couples the spatial resolution characteristics of an electron microscope with the temporal resolution of ultrafast Lasers. This first-of-its-kind instrument for imaging biological samples will be capable of sub-nanometer and nanosecond spatiotemporal resolution and will increase the combined attainable resolution five to eight orders of magnitude over current techniques. We further postulate that the resolution limiting effects observed during conventional cryo-EM imaging of biological samples arise from long-term specimen and electron beam interactions, and will be alleviated by using the Dynamic TEM with ultrafast imaging pulses. Mitigating these effects will result in improved three dimensional structure determination of macromolecular complexes. The impact of this research has the potential to elucidate new levels of understanding regarding microtubule and mitochondrial dynamics and the mechanisms involved with both normal and abnormal cellular processes. PUBLIC HEALTH RELEVANCE: Our work will provide the first high-resolution direct imaging of biological processes as they occur in real-time. These "live" movies will be acquired using a Dynamic Transmission Electron Microscope that combines characteristics of an atomic resolution electron microscope with ultrafast Lasers to study the structure of macromolecular complexes. This first-of-its-kind instrument will increase the spatial and temporal resolution for observing dynamic biological processes by five to eight orders of magnitude over current techniques.